Amyloidogenic protein aggregation is a key event in seemingly disparate biological phenomena, including those ranging from bacterial biofilms to neurodegenerative diseases. Intriguingly, amyloidogenic proteins do not form aggregates with a single 3D structure, but rather with several related yet distinct 3D structures that are linked to unique biological activities (akin to prion strains). How these structurally-unique aggregates mediate different toxic or beneficial activities is one of the most important questions in the protein aggregation field. Conformational antibodies specific for amyloidogenic aggregates are invaluable for investigating this intriguing problem and, more generally, for their potential use as disease-specific diagnostic and therapeutic agents. The utility of such antibodies stems from their ability to sensitively detect specific types of protein aggregates and selectively interfere with their biological activities. However, these types of antibodies are difficult to generate because of issues common to many antigens (immunodominant epitopes) and those specific to protein aggregates (multivalent, highly hydrophobic and, in some cases, kinetically unstable). The goals of this proposal are to develop structure-guided approaches for in vitro generation of antibodies against six classes of predicted conformational epitopes in amyloidogenic aggregates, and to use these antibodies to evaluate conformational differences between disease-associated aggregates formed in vivo. The predicted epitopes ? which include the leading candidates for generic antibody epitopes in oligomers and fibrils ? have not been specifically targeted or verified previously using monoclonal antibodies. We will primarily target a- synuclein (associated with Parkinson?s disease), which forms multiple types of oligomers and fibrils. These studies will address four key fundamental (Q1, Q2) and methodological (Q3, Q4) questions. 1) Which generic classes of predicted conformational antibody epitopes exist in oligomers and fibrils of a-synuclein and other amyloidogenic proteins? 2) Which protein-specific classes of predicted conformational epitopes exist in different structural (strain) variants of a-synuclein aggregates? 3) What are optimal design parameters for generating in vitro antibody libraries that maximize the likelihood of isolating antibodies with high specificity in addition to high affinity? 4) What are the most effective in vitro methods for targeting antibody libraries to pre- selected epitopes in protein aggregates in order to overcome common problems associated with immunodominant epitopes and poor antigen quality? These proposed studies are based on multiple discoveries in the Tessier lab: i) novel in vitro library design and sorting methods for isolating yeast-displayed antibodies with extremely high specificity; ii) bioinformatics methods for predicting antibody specificity; and iii) identification of multiple classes of conformational epitopes in Ab fibrils. These discoveries provide a strong basis for rationally generating antibodies against diverse types of amyloidogenic aggregates and for providing key insights into conformational differences between protein aggregates linked to unique human disorders.